DESCRIPTION: (applicant's description) The overall objective of this proposal is to develop an E. coli based vector system for the functional delivery of large genomic transgenes into human cells. Gene therapy holds great promise for the biomedical sciences in the treatment of genetic disease and cancer. Initial results with viral and liposome-based vector systems have been encouraging, but are limited by the relatively short length of DNA that can be delivered, with resultant variable levels and duration of gene expression. Delivering large genomic DNA-based transgenes, with sufficient surrounding genomic sequences to include not only the gene of interest but also endogenous promoters, introns and essential cis-acting elements, will display more accurate spatio-temporal expression compared to cDNA constructs. The efforts of the Human Genome Project have made large sequenced BAC and PAC clones containing human genes and surrounding genomic DNA readily available. However, lack of a suitable delivery method has hindered gene therapy studies using these valuable resources. Thus, the following three specific aims are designed to develop novel E. coli based gene therapy vectors capable of delivering large genomic clones. 1) An inducible homologous recombination system will be adapted to the E. coli DH10B to permit engineering of BACs. E. coli strain DH10B is recombination deficient (RecA-), making it the preferred vector for stably cloning large intact human genomic DNA into BACs. Providing several recombination proteins under control of an arabinose-depending promoter results in an inducible homologous recombination system. 2) E. coli DH10B will be made competent to invade mammalian cells and deliver large BACs, by expressing the Versinia pseudotuberculosis invasin gene and creating a deficiency in cell wall synthesis (dapA). 3)Human functional centromere DNA sequences will be engineered onto these BACs to provide mitotic stability to the transferred DNA in dividing cells. The human centromeric alpha satellite DNA has been shown to form de novo centromeres when introduced into human cells, creating human artificial chromosomes (HACs). However, these first generation HACs consist of greatly rearranged DNA, making them of limited use as gene expression vectors. The ability to engineer large BACs to contain human centromeric DNA and introduce then into human cells as HACs will overcome many of the limitations of previous HAC vectors. These studies propose to develop E. coli-based HAC vectors for human gene therapy that encompass novel approaches for gene delivery, accurate gene expression, and mitotic stability.